Nicotiana benthamiana 공장은 25 ° C에서 온도 제어 성장 챔버 재배하고 있습니다. 3~6주 오래된 식물의 완전 확장 건강한 단풍이 사용됩니다. 팁 : 더 나은 결과를 새로 신흥 잎을 사용하여 얻을 수 있습니다 1. Agrobacterium 과도 침투 프로토콜 : 1 일 <ol><li> 세포 죽음에 대한 assayed하는 유전자 (들)과 포함하는 벡터로 적절한 벡터를 포함하는 Agrobacterium tumefaciens (변형 LBA4404)의 글리세롤 주식과 리크 LB / Rifampicin (25 μg / ML) / Kanamycin (100 μg / ML) 한천 플레이트 제정 발기인 아래 거스 카세트. 항상 부정적인 컨트롤로 빈 벡터 제어를 포함합니다. </li><li> 28 ° C 2 일 품어. </li></ol> 3 일 <ol><li> / Rifampicin / Kanamycin 판은 이전에 줄무늬 각 LB에서 하나의 식민지와 Rifampicin (25 μg / ML)과 Kanamycin (100 μg / ML)를 포함하는 LB 2 ML의 예방. </li><li> 28 흔들어 ° C, 200 rpm으로 24 시간 최대 성장 밀도에 도달할 때까지 각 문화를 품다. </li></ol> 팁 : Clumping 때때로 경우 문화가 철저하게 진행하기 전에 (최대 pipetting 아래 IE) resuspended해야하는, 관찰됩니다. 일 사 <ol><li> 부화 후, 신선한 LB (적절한 항생제를 포함) 및 acetosyringone (최종 농도 25 μm의) 10 ML에 전체 볼륨을 높일 수 있습니다. </li><li> 또 다른 16 시간을위한 28 ° C에서 흔들어하여 각 문화를 품다. </li></ol> 5 일 <ol><li> 다음날 실온에서 10 분 4,000 XG 10 ML (acetosyringone를 추가하지 마십시오) 침투 매체 (10 MM MgSO 4 0.7 H 2 O, 9 MM MES, 산도 5.6) 및 원심 분리기를 추가하여 두 번 각각의 문화를 씻으십시오. </li><li> 최종 원심 분리 단계 후에, acetosyringone를 (100 μm의)이있는 5 ML의 침투 매체의 각 문화를 resuspend. </li><li> OD 600nm를 측정, 0.1과 0.9 사이에 측정 가능합니다. 팁 : 0.1만큼 낮은의 OD 600nm가 문제없이 사용되었습니다 그러나, 높은 ODS 더 일관성있는 결과를 얻을 수 있습니다. </li><li> 감염 Agrobacterium 문화를 준비하는보다 3 시간에 대한 문화를 품다. </li><li> 분석하는 유전자 (들)와 거스 카세트를 포함하는 문화와 1:1 비율에서 대조군 (Agrobacterium은 빈 벡터를 포함)를 포함하는 혼합 문화. </li><li> 1 ML 바늘 적은 주사기를 (그림 1)를 사용 새로 신흥 나뭇잎의 abaxial (아래) 측면을 침투. 팁 : 부정적인 제어 혼합 (빈 벡터 + 거스 카세트)와 같은 식물의 잎 반대에 assayed되도록 유전자를 (유전자 X + 거스 카세트)이있는 혼합물을 모두 침투. 각 치료 세중의 공장을 사용합니다. </li><li> 포스트 침투 소비세 3 일 거스 단백질 표현 나뭇잎과 분석에 침투. </li></ol> 2. Histochemical 거스 분석 일 6.25 <ol><li> excised 나뭇잎에 X - gluc 기판 매체에 침투 진공. </li><li> 하룻밤 상온이나 뚜렷한 파란색 얼룩이 나타날 때까지 어둠 속에 품어. </li></ol> 주 9 <ol><li> distillated 물에 씻어. </li><li> 엽록소가 제거 될 때까지 다시 증류수로 전송 후, 70 % 에탄올에 품어. 시각 거스 표현 수준을 평가. </li></ol> 3. Fluorometric 얼굴 분석 : 일 6.25 <ol><li> 액체 질소를 사용하여 절구에 침투 잎 디스크를 연마하여 전체 단백질을 분리. 거스 추출 버퍼 100 UL (50 MM NaPi 산도 7.0, 10 MM EDTA (에틸렌 다이아 민 테트라 초산), 0.1 % 트리톤 X - 100, 0.1 % N - lauroylsarcosine 나트륨 소금, β - 메르 캅 토 에탄올 (0.7 μl / ML) 얼음에 샘플을 유지하면서. 추가 </li><li> 4 ° C에서 5 분 1만4천그램에 현탁액을 원심과 총 가용성 단백질로 뜨는 가져가라. </li><li> 제조 업체의 지침에 따라 nanodrop를 사용하여 농도를 측정. </li><li> 거스 추출 버퍼를 사용하여 각 샘플에 대한 전체 가용성 단백질의 100 μg에 단백질 농도를 조정합니다. </li><li> 2 mm의 최종 농도 거스 추출 버퍼 준비 얼굴 기판 (4 methylumbelliferyl - β - D - glucuronide trihydrate)를 추가합니다. 샘플 당 100 μL (단백질 + 얼굴 기판)의 총 볼륨은 충분해야합니다. </li><li> 37 1 시간에 대한 반응을 품어 ° C. </li><li> 거스 중지 버퍼 (0.2 M 나 2 CO 3) 800 μl를 추가하여 반응을 중지합니다. </li><li> 365 NM 및 방출 파장 455 nm의의와 MU 표준 곡선에 값을 비교의 여기 파장에서 플레이트 판독기를 사용하여 형광을 측정합니다. nmol의 MU / μg 총 단백질 / 분 가용성으로 거스 활동의보고 수준. </li></ol> 4. 주식 및 솔루션 : <ol><li> Acetosyringone (1 M) 주식 (FW = 196.2 g) </하위&gt; - DMSO 5 ML에서 0.981 g </li><li> Kanamycin (100 MG / ML) 재고가 - DH 2 0 준비 - 필터 소독 </li><li> Rifampicin (25 MG / ML) 주식 - DMSO의 준비 </li><li> Luria - Bertani (LB) 액체 성장 미디어 (1L) 1% (W / V) bacto - tryptone, 10g 0.5 % (W / V) bacto - 효모 추출물, 5g 170 MM의 나트륨 염화물 10g 산도 - 7 </li><li> Acetosyringone (25 μm의) 1 ML의 최종 볼륨에 1 M의 주식 = 25 μl </li><li> 침투 매체 : (1L) 10 MM MgSO 4 0.7 DH 2 0 (FW = 246.48 g), 2.4648 g 9 밀리 MES (FW = 213.25 g), 1.91925 g 산도 - 5.6 </li><li> Acetosyringone (100 μm의) 1 ML의 최종 볼륨 1M 주식 = 100 μl </li><li> 거스 추출 버퍼 50 MM NaPi 산도 7.0, 10 MM EDTA (에틸렌 다이아 민 테트라 초산), 0.1 % 트리톤 X - 100, 0.1 % N - lauroylsarcosine 나트륨 소금, β - 메르 캅 토 에탄올 (0.7 μl / ML) </li><li> 2X 인산 버퍼 0.2M 아니 2 PO 4 0.2 M 나 2 HPO 4 산도 7 </li><li> X - Gluc 기판 솔루션 1 MG를 해산 5 브로모 -4 - 클로로 - 3 - 인돌릴 0.1 ML의 메탄올에 BD - glucuronide (X - Gluc). 1 ML 2X 인산 버퍼, ferricyanide 20 μl 0.1M의 칼륨, 10μl 트리톤 X - 100 10 % distillated 물을 850 μl를 추가합니다. </li></ol> 5. 대표 결과 : 세포 사망이 발생하면이 프로토콜에 따라, 거스 표현의 손실이 예상된다. 우리는 동시에 cyto - 보호 Arabidopsis BCL - 2 관련 athanoGene (BAG) 가족의 일원과 β - glucuronidase 리포터 유전자 (거스)을 표현했습니다. 우리는 공동 침투 N. benthamiana는 Agrobacterium 스트레인 LBA4404를 사용하여 35S 구동 가방 표현 카세트와 거스 벡터 pCAMBIA 2301로 떠납니다. 그림 2에 표시된 거스 얼룩에 눈에 띄는 증가는이 침투 다음과 같은 관찰되었다. 반대로, BCL - 2 가족의 알려진 프로 apoptotic 회원 백스가 사용되었을 때, 거스 얼룩의 표시 감소는 (그림 2) 관찰되었다. 이러한 경우 모두에서, 거스 표현식은 컨트롤에 비해 가시 달랐다. 그러나, 표현의 차이가 덜 분명하면 fluorometric 얼굴 assays은 거스 표현 quanitate을 수행할 수 있습니다. <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="그림 1"/> 그림 1. N.의 abaxial 측면의 Agrobacterium 혼합물의 침투의 예 benthamiana는 1 ML 바늘 적은 주사기를 사용하여 떠납니다. <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="그림 2"/> 그림 2. 거스 벡터는 N. 공동 표현했습니다 benthamiana는 안티 - 죽음의 유전자 및 세포 사망의 알려진 유도로 떠납니다. 거스 수준은 빈 벡터 제어 (거스 제어)에 비해했다.

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

관심 없음 충돌 선언하지 않습니다.

<p class="jove_content"&gt; 그것은 포유류의 시스템에서 일반적인 식물의 세포 사망 탐지 기술을 사용하는 것이 어렵습니다. 귀스 기자 제도와 함께, 우리는 세포 죽음 선수의 검출 및 분석을위한 식물을 기반으로, 중요한 방법을 제시한다. 이 방법은 라이브 세포가 발생하는 거스 표현에 필요한 그 단순한 사실을 활용합니다. 의미있는 결과와 repeatability를 보장하기 위해, 그것은 거스 카세트 및 assayed되도록 유전자를 숨?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> Rifampicin </td><td> VWR </td><td> IC19549001 </td><td> DMSO의 25mg/mL 주식 </td></tr><tr><td> 4&#39; - 히드록시 - 3 &#39;, 5&#39; - dimethoxyacetophenone (Acetosyringone) </td><td> VWR </td><td> TCD2666 </td><td> DMSO에서 0.981 g / ML (1M 주식) </td></tr><tr><td> 2 - (4 - morpholino) ethanesulfonic의 산성 일수화물 (MES) </td><td> VWR </td><td> EM - 6110 </td><td> 1.92 g / 침투 미디어 1 L를 만들기위한 물에 L </td></tr><tr><td> Bacto - tryptone </td><td> 어부 </td><td> BP1421 - 2 </td><td> 10g / LB 매체 1 L를 만들기위한 물에 L </td></tr><tr><td> Bacto - 효모 추출물 </td><td> VWR </td><td> EM1.03753.0500 </td><td> 5g / 물 L LB 매체 1 L을 만들기위한 </td></tr><tr><td> 나트륨 염화물 </td><td> VWR </td><td> EM - 7710 </td><td> 10g / LB 매체 1 L를 만들기위한 물에 L </td></tr><tr><td> 인산 나트륨 일염기의 일수화물 </td><td> VWR </td><td> MK - 7868-12 </td><td> 버퍼 NaPi의 50mM 만들기위한 물에 2.5G / L </td></tr><tr><td> 인산 나트륨 이염의 heptahydrate </td><td> VWR </td><td> EMD - SX0715 - 1 </td><td> 5g / 물 L 버퍼 NaPi의 50mM 만들기위한 </td></tr><tr><td> 마그네슘 황산의 heptahydrate </td><td> VWR </td><td> EM - MX0070 - 1 </td><td> 2.5 G / 침투 미디어 1 L를 만들기위한 물에 L </td></tr><tr><td> N - Lauroylsarcosine </td><td> VWR </td><td> TCL0151 - 500G </td><td> 0.1 % 거스 버퍼에 V / V </td></tr><tr><td> 5 - 브로모 -4 - 클로로 - 3 - indoxyl - 베타 - D - glucuronide cyclohexylammonium 소금 (X - gluc) </td><td> 골드 생명 공학 </td><td> G1281C </td><td> 메탄올 백퍼센트에 1mg/100uL </td></tr><tr><td> 4 Methylumbelliferyl - μ - D - glucuronide 하이드 레이트 (얼굴) </td><td> 시그마 </td><td> M5664 </td><td> 거스 추출 버퍼 100 UL 2 MM </td></tr><tr><td> 칼륨 ferrocyanide trihydrate </td><td> VWR </td><td> EM - PX1460 - 1 </td><td> X - gluc 기판 솔루션 100mM 주식 </td></tr><tr><td> β - Methylumbelliferone (MU) </td><td> 시그마 - 알드리치 </td><td> M1381 </td><td> MU 표준 곡선을 사용 거스 추출 버퍼 </td></tr><tr><td> 나트륨 탄산염 </td><td> VWR </td><td> EM - SX0395 - 11 </td><td> 거스 중지 버퍼를 만들기위한 물 0.2 M </td></tr></tbody></table>

a &#946;-glucuronidase (gus) based cell death assay

우리는 동시에 세포 죽음의 putative 긍정적이거나 부정적인 규제와 리포터 유전자, β - glucuronidase (거스)를 표현하는 소설 과도 공장 표현 시스템을 개발했습니다. 이 시스템에서는, N. benthamiana의 잎은 분석하는 유전자를 포함하는 35S 구동 표현 카세트 공동 침투되며, 거스 벡터 pCAMBIA 2301은 차량으로 Agrobacterium 스트레인 LBA4404를 사용합니다. 거스 표현식이 발생하는 라이브 세포가 필요하기 때문에이 마커 유전자가 세포 죽음의 긍정적인 규제와 함께 공동으로 표현 때 거스 활동의 손실이 예상된다. 마찬가지로, 거스 활동 안티 apoptotic 유전자는 벡터 제어에 비해 사용하는 경우 관찰 증가했다. 아래 그림과 같이, 우리는 성공적으로 모두 프로와 안티 죽음 플레이어를 분석하기 위해 실험실에서이 시스템을 사용하고 있습니다. 이들은 같은 백스와 같은 세포 사망의 포유 동물 inducers 알려진 안티 - apoptotic 공장 BCL - 2 관련 athanoGene (BAG) 가족뿐만 아니라를 포함. 또한, 우리는이 단백질의 가능한 포스트 translational 수정 / 정품 인증에 대한 단서를 제공할 수 단백질 내의 특정 truncations의 죽음 기능을 분석하기 위해이 시스템을 사용하고 있습니다. 여기, 우리는 특정 유전자의 죽음 기능을 조사하는 초기 단계로, 신속하고 민감한 식물을 기반으로 방법을 제시한다.

Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

A &#946;-glucuronidase GUS Based Cell Death Assay

일반적으로 이러한 DNA를 laddering 또는 TUNEL의 assays 같은 포유류의 시스템에서 사용되는 프로그램 세포 사망 assays은 종종 공장에서 재현하기가 어렵습니다. 귀스 기자 제도와 함께, 우리는 특정 유전자의 잠재적인 사형 속성을 분석하는 빠른, 식물 기반의 과도 분석을 제안합니다.

β - glucuronidase (거스)을 기반으로 세포를 파괴 분석

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

a-glucuronidase-gus-based-cell-death-assay

Research

JoVE Journal

Biology

A &#946;-glucuronidase (GUS) Based Cell Death Assay

28.0K 조회수.  Texas A&M University. 일반적으로 이러한 DNA를 laddering 또는 TUNEL의 assays 같은 포유류의 시스템에서 사용되는 프로그램 세포 사망 assays은 종종 공장에서 재현하기가 어렵습니다. 귀스 기자 제도와 함께, 우리는 특정 유전자의 잠재적인 사형 속성을 분석하는 빠른, 식물 기반의 과도 분석을 제안합니다.

비디오: A β-glucuronidase GUS Based Cell Death Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

루시페라제 기반 리포터 유전자 분석과 함께 막 Biogenesis 유학

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

연구

생물학

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

헬리 우스 진 건을 사용하여 양파 세포에서 단백질 - 단백질 상호 작용에 대한 Bimolecular 형광 Complementation (BiFC) 어세이

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

세포 죽음의 정확한 평가에 대한 수정 Annexin V / Propidium 이우의 Apoptosis 분석

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Cardiogenic 유도 효율을 Quantifying에 대한 수정 마우스 배아의 줄기 세포 기반 어세이

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

태아의 프로그래밍 셀 죽음을 감지 할 LysoTracker의 이용 및 배아 줄기 세포를 차별화

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

세포질 구성 요소를 식별 하는 간단한 형광 기반 기자 분석 결과 리 독 소에 필요한 체인 (RTA) 효 모에 인신 매매

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

SNARE 인신매매를 사용하여 재활용 내시경을 연구하고 분석하는 현미경 기반 분석

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

색색세포 기반 분석법을 사용하여 AAV에 대한 항체 중화를 검출하는 단계별 방법

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...